Myrac Aldehyde

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 September 2017 to 19 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 September 2017 to 19 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Dose selection rationale: The dietary levels used in this study (0, 3000, 6000 and 12000 ppm) were selected in conjunction with the Sponsor, following the completion of the preliminary toxicity study (Envigo Study Number PJ71TL). In that study there were no effects on clinical condition, body weight, food consumption, organ weights and macropathology findings at dietary concentrations of 3000, 6000 or 12000 ppm.
Therefore, a high dietary concentration of 12000 ppm was chosen for this study. The lowest dietary concentration of 3000 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 6000 ppm allowed evaluation of any concentration related trends of dietary concentrations.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 76 days old; Females: 83 to 90 days old.
- Weight at study initiation: Males: 325-397 g; Females: 246-310 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES: 03 January 2018 - 25 March 2018

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C). Formulations were prepared and used within the documented stability limits and met the following criteria: 4 days at ambient temperature, and 15 days frozen.

Details on mating procedure:

- Animals: Toxicity phase and Recovery phase males with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After a minimum of three weeks of treatment.
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of gestation.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 500 and 15000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.

Frequency of treatment:

Continuously

Dose / conc.:

0 ppm (analytical)

Remarks:

Group 1 (control)

Dose / conc.:

3 000 ppm (analytical)

Remarks:

Group 2 (low dose)

Dose / conc.:

6 000 ppm (analytical)

Remarks:

Group 3 (mid dose)

Dose / conc.:

12 000 ppm (analytical)

Remarks:

Group 4 (high dose)

No. of animals per sex per dose:

Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary levels used in this study (0, 3000, 6000 and 12000 ppm) were selected in conjunction with the Sponsor, following the completion of the preliminary toxicity study (Envigo Study Number PJ71TL). In that study there were no effects on clinical condition, body weight, food consumption, organ weights and macropathology findings at dietary concentrations of 3000, 6000 or 12000 ppm.
Therefore, a high dietary concentration of 12000 ppm was chosen for this study. The lowest dietary concentration of 3000 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 6000 ppm allowed evaluation of any concentration related trends of dietary concentrations.


- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.

- Post-exposure recovery period in satellite groups: 14 days

- Other: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Abnormal estrus cycle - 6 females; Ophthalmic abnormality - 1 female moved from Group 4 to Group 1.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter (including the recovery phase), and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Days 22 to 27), but recommenced for males on Day 27. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12of lactation).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4.
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group
- Anaesthetic used for blood collection: Yes, Animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Hematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Reticulocyte % (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt).
- Blood Chemistry parameters collected after overnight withdrawal of food on the Week 6: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile acids (Bi ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total prot), Albumin and Albumin/globulin ratio (A/G Ratio).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group;
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Blood pigments, Urobilinogen
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Thyroid Hormone Analysis
- Time schedule for examination

At termination: F0 males, All F0 Reproductive phase females

Day 4 of age - Offspring, two females selected for sampling per litter (if possible)
- one for T4 (serum)#
- one for TSH (plasma - optional analysis)
No females were selected for sampling if :
-The resultant live litter size would fall below ten pups
-The resultant number of live females would fall to less than three
-If only four female offspring were available within a litter but the overall litter size was >10, one female may be selected with priority given to the serum sample.
# priority given to serum sample for T4 analysis

Day 13 of age: Offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

Estrous Cycle
Wet smears:
Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to the Reproductive phase of the study.
- After pairing until mating.
- For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

Dry smears:
Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs (approximately three weeks).

Litter observations:

Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 final investigations were completed.
Reproductive phase females:
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Liver: First five toxicity phase males in Groups 2 and 3; All toxicity phase females in Groups 2 and 3; Recovery phase animals in Groups 1 and 4.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Postmortem examinations (offspring):

SACRIFICE
Time of necropsy:
Selected offspring for Day 4 thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
Method of sacrifice:
- Offspring- selected for thyroid hormone sampling on Day 4 or Day 13 of age: Decapitation
- Offspring - not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy, examined externally, and retained pending possible future examination.
- F1 offspring on Day 13 of age
Blood sampling required
All animals (but not including those selected for thyroid hormone analysis) were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined.

Statistics:

See "Any other information on materials and methods incl. tables"

Reproductive indices:

Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100
Gestation Length and Index: Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.
Gestation index was calculated for each group as:
- Gestation index (%) = (Number of live litters born / Number pregnant ) x 100

Offspring viability indices:

Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 13 of lactation / Number live offspring on Day 4 (after blood sampling)) x 100
Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs considered related to treatment with test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean bodyweight gains of females receiving 3000, 6000 and 12000 ppm over Days 1-8 before pairing were low when compared to Control and contributed to the low overall gain from Days 1-22; however overall bodyweight gains over Days 1-43 showed improvements in bodyweights at all dose levels. Bodyweights remained slightly low during gestation at 6000 and 12000 ppm and during lactation at 12000 ppm. The recovery period showed no effect of treatment for males but females previously exposed to 12000 ppm showed a higher bodyweight change during the recovery period (225% of Control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption for toxicity and recovery phase males before pairing receiving 3000, 6000 and 12000 ppm and toxicity, recovery and reproductive phase female before pairing receiving 6000 and 12000 ppm was lower than that of Control on Day 1 of treatment (Toxicity and recovery phase males: 92, 84 and 60%; Toxicity, recovery and reproductive females: 82 and 53%, respectively), with a dose-dependent decrease but was generally similar to that of the Controls intake in males and females thereafter.
Food consumption for males during the recovery phase was unaffected by treatment while females showed a higher mean food consumption during the recovery period (+24% compared to Control).
There was no significant effect on food consumption during the gestation phase.
During the lactation phase, females receiving 12000 ppm had statistically significantly low food consumption when compared with Control every day between Days 9 and 13 of lactation (83, 77, 74 and 78%, respectively). Food consumption at 3000 and 6000 ppm was unaffected during the lactation phase.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic lesions that were considered to be related to treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

The haematological examination of peripheral blood performed during Week 6 of treatment for males and females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. This included the statistically significant increase in hemoglobin concentration at 12000 ppm in males but within the historical control data range (13.1 to 16.8 L/L).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The biochemical examination performed during Week 6 of treatment for males and females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the statistically significant increase in sodium and potassium at 12000 ppm in males and the statistically significant increase in chloride at all dose levels in males with no dose-dependent trend and all within Historical Control Range (range: [sodium: 136 – 147 mmol/L], [potassium: 3.7 – 7.4 mmol/L], [Chloride: 95 – 105 mmol/L]).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis examination performed during Week 6 of treatment for males and females revealed increased urinary volume in males at all dose levels with statistical significance reached at 6000 and 12000 ppm, but without dose dependency - and well within historical control range (range: 3.0 – 16.2 mL).
At 6000 and 12000 ppm glucose, creatinine and chloride outputs were statistically significantly increased in males with no dose-dependent trend when compared with Control. No similar finding was evident in females. Potassium output was statistically significantly increased at all dose levels in males with a dose-dependent trend although this also was not evident in females.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The sensory reactivity and grip strength were unaffected by treatment.
The motor activity was unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Animals Killed After 6 Weeks of Treatment:
Changes related to treatment with tets item were seen in the liver.
In animals, treated with 12000 ppm 3-cyclohexene-1-carboxaldehyde multiconstituent in the diet, minimal hepatocellular hypertrophy was identified in all males and females. Hypertrophy was generally diffuse in the females but more prominent in the periportal to midzonal regions in males. The incidence and distribution of all other findings were considered to be unrelated to treatment.
- Animals Killed After 2 Weeks of Recovery:
Livers and tissues with abnormalities were examined. There were no significant findings in the livers of animals following a 14-day recovery period after treatment with 12000 ppm 3-cyclohexene-1-carboxaldehyde multiconstituent in the diet. All histological changes seen in tissues identified with abnormalities were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
Thyroxine (T4): All samples taken from adult males at termination and Day 13 of age male and female offspring from the animals in the control group (Group 1) had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. However, lower concentrations of T4 were found in samples taken from adult males at termination and Day 13 of age male and female offspring from the animals in the 3000 to 12000 ppm groups and statistical significance was achieved in all treated groups. A decrease in the T4 concentration found is observed with increasing dose concentrations. This effect is more pronounced in Day 13 offspring animals (for both genders) than in the terminal male animals.
Thyroid stimulating hormone (TSH):TSH concentrations were found to be statistically significantly higher for Male offspring (F1 generation) dosed with 12000 ppm of 3-cyclohexene-1-carboxaldehyde multiconstituent compared to the control group but without dose-dependent increase and with one animal with a very high value. Although statistical significance was not achieved in the male or female (F1 generation) offspring of other treated groups, generally these groups had higher levels of plasma TSH concentrations but not dose-related, when compared to Control. There was no difference in the concentration of adult males, when compared to Control.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Prior to treatment all females had regular estrus cycles of 4 to 5 days with the exception of one Group 2 toxicity phase female who had an irregular cycle.
During treatment for the reproductive phase, 9 Control females, 8 females receiving 3000 ppm, 7 females receiving 6000 ppm and 7 females receiving 12000 ppm had regular 4 to 5 day estrus cycles. One female in all treated groups had an irregular cycle, one female receiving 6000 ppm had extended estrus and one female in Control, 3000 and 6000 ppm, and two females receiving 12000 ppm were acyclic.
At the end of treatment, all non-mated females showed regular estrous cycles and all reproductive phase females showed diestrus.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Pre-coital interval, mating performance, gestation length and gestation index was unaffected by treatment.
The live birth index (%) at 12000 ppm (91.8%) was low when compared to Control and was outside of Historical Control Data (range 96.2 – 100%) and is therefore considered adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

6 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical condition of the offspring, ano-genital distance on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Male offspring mean bodyweights were statistically significantly low on Day 4 and 13 of age at 12000 ppm, contributing to a statistically low overall bodyweight gain over Days 1-13 when compared to Control.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Ano-genital distance in male and female offspring was unaffected by treatment.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings in decedent offspring, or offspring at termination on Day 13 of age, that were considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

6 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive performance of parents (live birth index)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation Analysis:

The mean concentrations for all groups were within the acceptance limits of +10/-15% with the exception of Group 3 for the final week of treatment which was -17.3% from nominal. The recoveries for each occasion remained within ±10% of the mean recovery found during validation showing the continued accuracy of the method.

Achieved Dose:

Mean achieved doses for toxicity, recovery phase and reproductive phase before pairing, for males, were 174, 357 and 714 mg/kg bw/day at 3000, 6000 and 12000 ppm, respectively.

Mean achieved doses for toxicity, recovery phase and reproductive phase before pairing, for females, were 189, 365 and 744 mg/kg bw/day at 3000, 6000 and 12000 ppm, respectively.

Mean Achieved doses for females at 3000, 6000 and 12000 ppm were respectively 190, 361 and 761mg/kg bw/day during lactation and, respectively 431, 809, 1480 mg/kg bw/day during lactation.

Conclusions:

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of test item for systemic toxicity was 12000 ppm (mean achieved doses of 714 mg/kg bw/day for males, 744 mg/kg bw/day for toxicity phase females, 761 mg/kg bw/day for females during gestation and 1480 mg/kg bw/day for females during lactation). The low live birth index (%) at 12000 ppm was considered as adverse, as the value was below the historical control data values, therefore the NOAEL for reproductive/developmental toxicity is considered to be 6000 ppm (gestation: 361 mg/kg bw/day; lactation: 809 mg/kg bw/day).

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 3000, 6000 and 12000 ppm.

 

Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks. Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, urinalysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The mean concentrations of test item for all groups were within the acceptance limits of +10/-15% with the exception of Group 3 for the final week of treatment which was -17.3% from nominal. The recoveries for each occasion remained within ±10% of the mean recovery found during validation showing the continued accuracy of the method.

Dietary administration of test item at levels of 3000, 6000 and 12000 ppm was generally well tolerated. There was no premature deaths and no adverse effect attributed to treatment on clinical condition, sensory reactivity and grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, ophthalmoscopy, hematology, blood chemistry, urinalysis or macropathology.

Achieved dose, generally maintained the intervals between dietary concentrations. During lactation, achieved doses were higher, reflecting the increased physiological demand on the dams.

Mean bodyweight gains of females receiving 3000, 6000 and 12000 ppm over Days 1-8 before pairing were low when compared to Control and contributed to the low overall gain from Days 1-22; however overall bodyweight gains over Days 1-43 showed improvements in bodyweights at all dose levels. Bodyweights remained slightly low during gestation at 6000 and 12000 ppm and during lactation at 12000 ppm. The recovery period showed no effect of treatment for males but females previously exposed to 12000 ppm showed a higher bodyweight change during the recovery period (225% of Control).

The effects observed on bodyweights at the beginning of treatment were associated with a dose-dependent decrease in food consumption on day 1 of treatment for males and females before pairing, probably due to the low palatability of the preparations; however food consumption was generally similar to that of Controls thereafter except from Day 9 to Day 13 of lactation where food consumption was statistically significantly low. Conversely, females previously exposed to 12000 ppm showed a higher mean food consumption during the recovery period (+23% compared to Control).

 

Macroscopic examination performed after six weeks of treatment, two weeks of recovery or following completion of the reproductive phase, revealed no test-item related lesions. The incidence and distribution of all findings were consistent with the common background seen in Sprague-Dawley rats at these laboratories. 

Animals exposed to 12000 ppm 3-cyclohexene-1-carboxaldehyde multiconstituent in the diet showed hepatocellular hypertrophy in the liver which correlated with increased organ weight. After two weeks of recovery there was no difference in the organ weight of the liver and no evidence of hepatocellular hypertrophy on histopathological examination suggesting complete reversibility of the finding. Hepatocellular hypertrophy is a common finding following exposure to a variety of chemical agents. It is often, but not exclusively, correlated to hepatic microsomal enzyme induction and considered as an adaptive finding which commonly reverses when treatment is stopped (Greaves 2011).

This study included a screen for reproductive/developmental effects. Estrous cycles, pre‑coital interval, mating performance, fertility and gestation length were unaffected by treatment. There were also no effects on post implantation survival or lactation index (%), or on the ratio of males to females. However, the live birth index (%) at 12000 ppm (91.8%) was low when compared to Control and was outside of Historical Control Data (range 96.2 – 100%) and is therefore considered adverse. On Day 4, the viability index at 12000 ppm was also low, however the value was marginally within Historical Control Data; there was clear increase in pup mortality at 12000 ppm between birth and Day 4 of age with 24 pups dying/killed for welfare reasons compared with 6 control pups. The clinical condition of the offspring, ano-genital distance on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment. Male offspring mean bodyweights were statistically significantly low on Day 4 and 13 of age at 12000 ppm, contributing to a statistically low overall bodyweight gain over Days 1-13 when compared to Control.

The measurement of the hormone Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) in adult reproductive males and in Day 13 offspring was also included. Non-adverse changes in T4 and TSH are associated with a dose-related increase in liver weights in adult animals and an hypertrophy of the liver in the high dose animals. There is evidence that thyroid hormone perturbations mediated via increased excretion are generally a rodent-specific phenomenon due to the significantly greater sensitivity of hepatic enzyme induction in rodents versus humans and the much greater turnover of thyroid hormones in rodents in comparison to humans (McClean, 1995; Colnot and Dekant, 2017). Because some developmental stages (e.g. gestational and neo-natal) are particularly sensitive to endocrine effects, an external examination of all offspring was conducted, measurement of the ano-genital distance of offspring on Day 1 of age was undertaken and nipple counts for male offspring on Day 13 of age were assessed.  The microscopic examination of the reproductive organs was unremarkable even after up to 13 weeks of exposure in males. All offspring were macroscopically ‘normal’, in particular no effects were seen on the external genitalia. Ano‑genital distance and male nipple count were not adversely affected by treatment. In the context of this study, the weight of evidence supports that 3-cyclohexene-1-carboxaldehyde multiconstituent has no effect on endocrine systems.  

 

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of test item for systemic toxicity was 12000 ppm (mean achieved doses of 714 mg/kg bw/day for males, 744 mg/kg bw/day for toxicity phase females, 761mg/kg bw/day for females during gestation and 1480 mg/kg bw/day for females during lactation). The low live birth index (%) at 12000 ppm was considered as adverse, as the value was below the historical control data values, therefore the NOAEL for reproductive/developmental toxicity is considered to be 6000 ppm (gestation: 361 mg/kg bw/day; lactation: 809 mg/kg bw/day).

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Preliminary study to select a suitable high dose for a subsequent OECD 422 combined toxicity and reproductive/development screening study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 November 2017 to 26 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The preliminary study was not designed to meet any particular regulatory requirements, used as range-finder experiment for OECD 422 screening test performed in GLP laboratory.

Qualifier:

no guideline available

Principles of method if other than guideline:

Due to the preliminary nature of this study, no specific regulations or guidelines are
applicable. The study was not designed to meet any particular regulatory requirements.

GLP compliance:

no

Remarks:

study was conducted in a GLP facility, however no claim of GLP compliance was intended/made for this study.

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain, accepted by regulatory agencies, was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males: 69-76 days; Females: 83-90 days
- Weight at study initiation: Males: 328-383 g; Females: 223-271 g
- Housing: Four animals of the same sex were housed per cage of polycarbonate body with a stainless steel mesh lid. Wood based bedding which was changed at appropriate intervals each week.
- Diet: SDS VRF1 Certified powdered diet; ad libitum (removed overnight before blood sampling for blood chemistry collection/investigations).
- Water: Potable water from the public supply; ad libitum
- Acclimation period: Six days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24ºC
- Humidity: 40-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours light / 12 hours dark
- IN-LIFE DATES: 15 November to 12 December 2017.

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate a condition of potential human exposure.

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: A correction factor was not required.
- Method of preparation:
- Rate of preparation of diet (frequency): Weekly; formulations were prepared and used within the documented stability limits and met the following criteria: 4 days at ambient temperature, and 15 days frozen.
- Mixing appropriate amounts with (Type of food): On each occasion of the preparation of the premix the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture was stirred. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred as well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer. For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Storage temperature of food: Frozen (-10 to -30°C) for up to 15 days and thawed at ambient temperature (15-25°C) immediately before use.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analysis of the formulated diets was performed on this study, however, samples of the first preparations were stored at -20 °C.

Duration of treatment / exposure:

21 days

Frequency of treatment:

Continuously

Dose / conc.:

0 ppm

Remarks:

Group 1 (Control)

Dose / conc.:

5 000 ppm

Remarks:

Group 2 (Low dose)

Dose / conc.:

10 000 ppm

Remarks:

Group 3 (Mid dose)

Dose / conc.:

15 000 ppm

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION RATIONALE:
The dietary concentrations used in this study (0, 5000, 10000, and 15000 ppm) were selected in conjunction with the Sponsor based on:
- In a recent acute oral toxicity study conducted according to OECD guideline 423 in rats, the LD50 of the test item was found to be higher than 2000 mg/kg body weight.

Based on these results, 15000 ppm (equivalent to 1000 mg/kg/bw/day) was selected as the highest dietary concentration in order to assess the palatability and the systemic toxicity of this test item. The intermediate dietary concentration of 10000 ppm was chosen to enable the identification of a high dietary concentration for the OECD 422 study. The low dietary concentration of 5000 ppm was expected to be a no effect level.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed on each animal on Days -3, 1, 4, 8, 11, 15, 18 and 21 on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily throughout the study from Day -3 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily throughout the study from Day -3.
- The group mean achieved dose for each sex, expressed as mg/kg bw/day, was calculated for each week from the nominal dietary test item concentration, food consumption and body weight data.
The following formula was used:
Achieved dose (mg/kg/day) = Mean food consumed (g/animal) * ppm test substance / Mid period body weight (g)

The group mean mid period body weight was used in the formula. Overall group mean values were calculated from the weekly group mean values presented.

WATER CONSUMPTION: No
- Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at week 3
- Parameters examined: Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin by chemical assay (Alb). Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

ESTROUS CYCLES: Yes
- Time schedule: Daily smears were taken for 21 days, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the estrous cycle.

Sacrifice and pathology:

GROSS PATHOLOGY: All animals were subject to a detailed necropsy. Only the thoracic and abdominal cavities were opened. The cranial cavity was not opened as there were no observations during the study to indicate a possible neurotoxic action. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed individually and presentated in the table 7.5.1/1. Requisite organs were weighed for animals killed at the scheduled interval.

HISTOPATHOLOGY: No; but tissues with macroscopic abnormalities were preserved for microscopic examinations in 10% neutral buffered formalin for microscopic examination (except testes: In modified Davidson’s fluid).

Other examinations:

None

Statistics:

Summary statistics (e.g. means and standard deviations) were calculated from computer-stored individual raw data. No statistical analysis of the data was performed on this study.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related changes in clinical condition during the study.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Overall body weight gain of males receiving 15000 ppm was low (60% of Control). Overall body weight gain in all other treated groups were unaffected by treatment. Any differences seen in the females was attributable to the performance of one female (4F 104) in the control group which experienced body weight loss and whose body weight performance was dissimilar to the other females in the group. Mean body weight loss of males receiving the test item at 10000 and 15000 ppm was greater than the Controls during Days 1 to 2 of treatment (-9g and -16g, respectively). The bodyweight gain of males receiving 15000 ppm was slightly lower to that of the Control thereafter (79% of Control from Days 3-22 of treatment).
- Following the start of treatment, females receiving 10000 or 15000 ppm showed a higher mean weight loss on Day 2 of study (-11g and -16g, respectively) compared with a mean weight loss of -3g in Controls for the same period. Though this was considered attributable to the palatability of the basal diet with corn oil and/or the basal diet with corn oil and test item. The body weight gain of females receiving 10000 or 15000 ppm was greater than Controls thereafter (166% and 250% of Control, respectively, from Days 3-22 of treatment).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Overall (Days 1 to 21) food intake of males receiving 15000 ppm was marginally low (92%), when compared with Control: this was heavily influenced by the low food on Day 1 (48% of Control). Food intake on Day 1 was also slightly low in males receiving 10000 ppm (72%).
- Food intake was low during the first two days in females receiving 10000 or 15000ppm (58 or 53% of Control, respectively).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Water consumption was unaffected by treatment.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Blood chemistry analyses for males revealed some differences from Controls, including mean alkaline phosphatase levels which were greater than that of the Control for all groups of males and females receiving all doses of test item (121, 111 or 136% and 134, 164 or 186% of Control, respectively), Mean bile acid concentrations were higher than that of the Control for males and females receiving all doses of test item (323, 407 or 323% and 236 or 747% of Control, respectively).

-All other differences from Control were minor or were confined to one sex and were therefore attributed to normal biological variation. These included, statistically significantly high alanine aminotransferase activity in males receiving 15000 ppm (127 or 116% of Control, respectively) and statistically significantly high creatinine concentration in males receiving 10000 or 15000 ppm (117 or 111% of Control, respectively). Calcium concentrations were statistically significantly low in males receiving 10000 or 15000 ppm (96 or 97% of Control, respectively) and albumin concentration was statistically significantly high in males receiving 15000 ppm (109% of Control).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight-adjusted liver weights were slightly higher than that of Controls for all groups of treated males and for females receiving 10000 or 15000 ppm. Adjusted kidney weights were also slightly high for males receiving 10000 or 15000 ppm (119 or 118% of Control, respectively) and females receiving 15000 ppm (112% of Control).
All other inter-group differences were minor and/or lacked dose-relationship and were therefore attributed to normal biological variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One female (No. 105) treated at 10000 ppm had adhesions in the abdominal cavity and thickening of the capsule of the spleen that were not evident at 15000 ppm and was therefore considered not to be toxicologically significant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Two females (No’s 110 and 111) receiving 10000 ppm showed irregular estrous cycles that was not evident in females receiving 5000 or 15000 ppm. Irregular length of estrous cycle is often seen in some animals and this was therefore considered not to be biologically relevant or toxicologically significant.

Details on results:

No additional details

Dose descriptor:

dose level: 5000, 10000 or 15000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: dietary administration of test item for 21 days at 5000, 10000 or 15000 ppm was well tolerated in rats. It is considered that the high dose level on the subsequent OECD 422 study should be 12000 ppm.

Critical effects observed:

no

None

Conclusions:

Under the test conditions, the administration of Test item to CD rats by dietary administration for 21 days at dietary levels of 5000, 10000 or 15000 ppm was well tolerated. No animals died and the general clinical condition of the animals was unaffected by treatment (water consumption, estrous cycles or macropathology). At 15000 ppm, overall body weight gain and food intake on Day 1 in males receiving15000 ppm were low. Food intake was also slightly low on Day 1 in males receiving 10000 ppm and, low on days 1 and 2 in females receiving 15000 or 10000 ppm. This was attributed to the palatability of the basal diet with the addition of corn oil, as the Controls showed a similar effect. Biochemical investigation of the blood plasma in the final week of treatment indicated high alkaline phosphatase activity, high bile acid concentration and low triglyceride concentration in males and females receiving 5000, 10000 or 15000 ppm. It is considered that these changes may indicate a mild toxic insult on the liver since these changes corresponded with high liver weights in males at these doses or in females at 10000 or 15000 ppm. The increased kidney weights in males at 10000 or 15000 ppm and in females at 15000 ppm did not correlate with any changes in biochemical parameters assessed or any findings at macroscopic examination.Broadly similar small mean body weight losses were evident in both the Control and all treated groups for one day after the first feed. It is considered that based on low food intake in males at 15000 ppm along with the increase of liver and kidney weights, the high dose level on the subsequent OECD 422 study should be 12000 ppm. Therefore, the doses to be tested in the main OECD 422 study were 3000, 6000, 12000 ppm.

Executive summary:

In a 21-day oral dietary toxicity study, conducted to select a suitable high dose for a subsequent OECD 422 combined toxicity and reproductive/development screening study, four groups of Crl:CD(SD) rats (4/sex/dose) received the Test item at dietary concentrations of 0 (vehicle, basal diet, with added corn oil), 5000, 10000 and 15000 ppm (equivalent to mean achieved doses of 305, 610 and 853 mg/kg bw/day in males and 328, 613 and 958 mg/kg bw/day in females, respectively). During the study, clinical condition, body weight, food consumption, visual water consumption, estrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken.

The administration of 3-cyclohexene-1-carboxaldehyde multiconstituent to Crl:CD (SD) rats by dietary administration for 21 days at dietary levels of 5000, 10000 or 15000 ppm was well tolerated. No animals died and the clinical condition of the animals was satisfactory. The effects on bodyweight and food consumption at 15000 ppm at the start of treatment, from which the animals quickly recovered, were considered likely to be related to the palatability

of test item 3-cyclohexene-1-carboxaldehyde multiconstituent. Increase in liver (males at all dietary concentrations and females at 10000 or 15000 ppm) or kidney weights (males at 10000 or 15000 ppm and females at 15000 ppm were seen. There were no other findings attributable to treatment seen at macroscopic examination.

Under the test conditions, it is considered that based on low food intake in males at 15000 ppm along with the increase of liver and kidney weights, the high dose level on the subsequent OECD 422 study should be 12000 ppm. Therefore, the doses to be tested in the main OECD 422 study were 3000, 6000, 12000 ppm.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

253-617-4

EC Name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

37677-14-8

Molecular formula:

C13H20O

IUPAC Name:

4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

Constituent 2

Reference substance name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

257-943-8

EC Name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

52475-89-5

Molecular formula:

C13H20O

IUPAC Name:

3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No. 190826
Purity: 99.6% (sum of the two main constituents)
Name of the test item (as cited in the study report): 3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENT
Physical state: colourless-slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 05 September 2018

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 76 days old; Females: 83 to 90 days old.
- Weight at study initiation: Males: 325-397 g; Females: 246-310 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES: 03 January 2018 - 25 March 2018

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C). Formulations were prepared and used within the documented stability limits and met the following criteria: 4 days at ambient temperature, and 15 days frozen.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 500 and 15000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

Group 2 (low dose)

Doses / concentrations 3

Dose / conc.:

6 000 ppm

Remarks:

Group 3 (mid dose)

Doses / concentrations 4

Dose / conc.:

12 000 ppm

Remarks:

Group 4 (high dose)

No. of animals per sex per dose:

Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary levels used in this study (0, 3000, 6000 and 12000 ppm) were selected in conjunction with the Sponsor, following the completion of the preliminary toxicity study (Envigo Study Number PJ71TL). In that study there were no effects on clinical condition, body weight, food consumption, organ weights and macropathology findings at dietary concentrations of 3000, 6000 or 12000 ppm.
Therefore, a high dietary concentration of 12000 ppm was chosen for this study. The lowest dietary concentration of 3000 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 6000 ppm allowed evaluation of any concentration related trends of dietary concentrations.


- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.

- Post-exposure recovery period in satellite groups: 14 days

- Other: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Abnormal estrus cycle - 6 females; Ophthalmic abnormality - 1 female moved from Group 4 to Group 1.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter (including the recovery phase), and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Days 22 to 27), but recommenced for males on Day 27. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12of lactation).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4.
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group
- Anaesthetic used for blood collection: Yes, Animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Hematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Reticulocyte % (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt).
- Blood Chemistry parameters collected after overnight withdrawal of food on the Week 6: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile acids (Bi ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total prot), Albumin and Albumin/globulin ratio (A/G Ratio).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group;
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Blood pigments, Urobilinogen
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 final investigations were completed.
Reproductive phase females:
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Liver: First five toxicity phase males in Groups 2 and 3; All toxicity phase females in Groups 2 and 3; Recovery phase animals in Groups 1 and 4.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Other examinations:

Thyroid Hormone Analysis
- Time schedule for examination

At termination: F0 males, All F0 Reproductive phase females

Statistics:

See "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Dietary administration of 3-cyclohexene-1-carboxaldehyde multiconstituent at levels of 3000, 6000 and 12000 ppm was generally well tolerated and showed no test-item related changes in clinical condition.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean bodyweight gains of females receiving 3000, 6000 and 12000 ppm over Days 1-8 before pairing were low when compared to Control and contributed to the low overall gain from Days 1-22; however overall bodyweight gains over Days 1-43 showed improvements in bodyweights at all dose levels. Bodyweights remained slightly low during gestation at 6000 and 12000 ppm and during lactation at 12000 ppm. The recovery period showed no effect of treatment for males but females previously exposed to 12000 ppm showed a higher bodyweight change during the recovery period (225% of Control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption for toxicity and recovery phase males before pairing receiving 3000, 6000 and 12000 ppm and toxicity, recovery and reproductive phase female before pairing receiving 6000 and 12000 ppm was lower than that of Control on Day 1 of treatment (Toxicity and recovery phase males: 92, 84 and 60%; Toxicity, recovery and reproductive females: 82 and 53%, respectively), with a dose-dependent decrease but was generally similar to that of the Controls intake in males and females thereafter.
Food consumption for males during the recovery phase was unaffected by treatment while females showed a higher mean food consumption during the recovery period (+24% compared to Control).
There was no significant effect on food consumption during the gestation phase.
During the lactation phase, females receiving 12000 ppm had statistically significantly low food consumption when compared with Control every day between Days 9 and 13 of lactation (83, 77, 74 and 78%, respectively). Food consumption at 3000 and 6000 ppm was unaffected during the lactation phase.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic lesions that were considered to be related to treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

The haematological examination of peripheral blood performed during Week 6 of treatment for males and females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. This included the statistically significant increase in hemoglobin concentration at 12000 ppm in males but within the historical control data range (13.1 to 16.8 L/L).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The biochemical examination performed during Week 6 of treatment for males and females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the statistically significant increase in sodium and potassium at 12000 ppm in males and the statistically significant increase in chloride at all dose levels in males with no dose-dependent trend and all within Historical Control Range (range: [sodium: 136 – 147 mmol/L], [potassium: 3.7 – 7.4 mmol/L], [Chloride: 95 – 105 mmol/L]).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis examination performed during Week 6 of treatment for males and females revealed increased urinary volume in males at all dose levels with statistical significance reached at 6000 and 12000 ppm, but without dose dependency - and well within historical control range (range: 3.0 – 16.2 mL).
At 6000 and 12000 ppm glucose, creatinine and chloride outputs were statistically significantly increased in males with no dose-dependent trend when compared with Control. No similar finding was evident in females. Potassium output was statistically significantly increased at all dose levels in males with a dose-dependent trend although this also was not evident in females.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The sensory reactivity and grip strength were unaffected by treatment.
The motor activity was unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following six weeks of treatment, adjusted liver weight was statistically significantly high in males (126%) and females (127%) at 12000 ppm when compared with Control.
All other differences in organ weights were minor, or evident in a single sex and were therefore considered unaffected by treatment. These included the statistically significant decrease in adjusted adrenal weight, and the statistically significant increase in adjusted spleen and thymus weights in females receiving 12000 ppm – all, except for thymus weights, were within the historical control range (range [adjusted]: [adrenals: 0.076 0.096 g], [thymus: 0.120 – 0.214 g], [Spleen: 0.513 – 0.708 g])
Following two weeks respite from treatment at 12000 ppm, absolute and adjusted liver weights were comparable with Control.
On Day 13 of lactation, adjusted uterus, cervix and oviduct weights saw a dose-dependent decrease in weight with statistical significance reached at 6000 and 12000 ppm in females (87% and 83% at 6000 and 12000 ppm, respectively) which was slightly outside the Historical Control Range (range [adjusted] values: 0.391 – 0.565 g).

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed revealed no test item related lesions in all groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Animals Killed After 6 Weeks of Treatment:
Changes related to treatment with tets item were seen in the liver.
In animals, treated with 12000 ppm 3-cyclohexene-1-carboxaldehyde multiconstituent in the diet, minimal hepatocellular hypertrophy was identified in all males and females. Hypertrophy was generally diffuse in the females but more prominent in the periportal to midzonal regions in males. The incidence and distribution of all other findings were considered to be unrelated to treatment.
- Animals Killed After 2 Weeks of Recovery:
Livers and tissues with abnormalities were examined. There were no significant findings in the livers of animals following a 14-day recovery period after treatment with 12000 ppm 3-cyclohexene-1-carboxaldehyde multiconstituent in the diet. All histological changes seen in tissues identified with abnormalities were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
Thyroxine (T4): All samples taken from adult males at termination and Day 13 of age male and female offspring from the animals in the control group (Group 1) had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. However, lower concentrations of T4 were found in samples taken from adult males at termination and Day 13 of age male and female offspring from the animals in the 3000 to 12000 ppm groups and statistical significance was achieved in all treated groups. A decrease in the T4 concentration found is observed with increasing dose concentrations. This effect is more pronounced in Day 13 offspring animals (for both genders) than in the terminal male animals.
Thyroid stimulating hormone (TSH):TSH concentrations were found to be statistically significantly higher for Male offspring (F1 generation) dosed with 12000 ppm of 3-cyclohexene-1-carboxaldehyde multiconstituent compared to the control group but without dose-dependent increase and with one animal with a very high value. Although statistical significance was not achieved in the male or female (F1 generation) offspring of other treated groups, generally these groups had higher levels of plasma TSH concentrations but not dose-related, when compared to Control. There was no difference in the concentration of adult males, when compared to Control.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Formulation Analysis:

The mean concentrations for all groups were within the acceptance limits of +10/-15% with the exception of Group 3 for the final week of treatment which was -17.3% from nominal. The recoveries for each occasion remained within ±10% of the mean recovery found during validation showing the continued accuracy of the method.

Achieved Dose:

Mean achieved doses for toxicity, recovery phase and reproductive phase before pairing, for males, were 174, 357 and 714 mg/kg bw/day at 3000, 6000 and 12000 ppm, respectively.

Mean achieved doses for toxicity, recovery phase and reproductive phase before pairing, for females, were 189, 365 and 744 mg/kg bw/day at 3000, 6000 and 12000 ppm, respectively.

Mean Achieved doses for females at 3000, 6000 and 12000 ppm were respectively 190, 361 and 761 mg/kg bw/day during lactation and, respectively 431, 809, 1480 mg/kg bw/day during lactation.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of test item for systemic toxicity was 12000 ppm (mean achieved doses of 714 mg/kg bw/day for males, 744 mg/kg bw/day for toxicity phase females, 761mg/kg bw/day for females during gestation and 1480 mg/kg bw/day for females during lactation).

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 3000, 6000 and 12000 ppm.

 

Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks. Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, urinalysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The mean concentrations of test item for all groups were within the acceptance limits of +10/-15% with the exception of Group 3 for the final week of treatment which was -17.3% from nominal. The recoveries for each occasion remained within ±10% of the mean recovery found during validation showing the continued accuracy of the method.

Dietary administration of test item at levels of 3000, 6000 and 12000 ppm was generally well tolerated. There was no premature deaths and no adverse effect attributed to treatment on clinical condition, sensory reactivity and grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, ophthalmoscopy, hematology, blood chemistry, urinalysis or macropathology.

Achieved dose, generally maintained the intervals between dietary concentrations. During lactation, achieved doses were higher, reflecting the increased physiological demand on the dams.

Mean bodyweight gains of females receiving 3000, 6000 and 12000 ppm over Days 1-8 before pairing were low when compared to Control and contributed to the low overall gain from Days 1-22; however overall bodyweight gains over Days 1‑43 showed improvements in bodyweights at all dose levels. Bodyweights remained slightly low during gestation at 6000 and 12000 ppm and during lactation at 12000 ppm. The recovery period showed no effect of treatment for males but females previously exposed to 12000 ppm showed a higher bodyweight change during the recovery period (225% of Control).

The effects observed on bodyweights at the beginning of treatment were associated with a dose-dependent decrease in food consumption on day 1 of treatment for males and females before pairing, probably due to the low palatability of the preparations; however food consumption was generally similar to that of Controls thereafter except from Day 9 to Day 13 of lactation where food consumption was statistically significantly low. Conversely, females previously exposed to 12000 ppm showed a higher mean food consumption during the recovery period (+23% compared to Control).

 

Macroscopic examination performed after six weeks of treatment, two weeks of recovery or following completion of the reproductive phase, revealed no test-item related lesions. The incidence and distribution of all findings were consistent with the common background seen in Sprague-Dawley rats at these laboratories. 

Animals exposed to 12000 ppm 3-cyclohexene-1-carboxaldehyde multiconstituent in the diet showed hepatocellular hypertrophy in the liver which correlated with increased organ weight. After two weeks of recovery there was no difference in the organ weight of the liver and no evidence of hepatocellular hypertrophy on histopathological examination suggesting complete reversibility of the finding. Hepatocellular hypertrophy is a common finding following exposure to a variety of chemical agents. It is often, but not exclusively, correlated to hepatic microsomal enzyme induction and considered as an adaptive finding which commonly reverses when treatment is stopped (Greaves 2011).

The measurement of the hormone Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) in adult reproductive males and in Day 13 offspring was also included. Non-adverse changes in T4 and TSH are associated with a dose-related increase in liver weights in adult animals and an hypertrophy of the liver in the high dose animals. There is evidence that thyroid hormone perturbations mediated via increased excretion are generally a rodent-specific phenomenon due to the significantly greater sensitivity of hepatic enzyme induction in rodents versus humans and the much greater turnover of thyroid hormones in rodents in comparison to humans (McClean, 1995; Colnot and Dekant, 2017). Because some developmental stages (e.g. gestational and neo-natal) are particularly sensitive to endocrine effects, an external examination of all offspring was conducted, measurement of the ano-genital distance of offspring on Day 1 of age was undertaken and nipple counts for male offspring on Day 13 of age were assessed.  The microscopic examination of the reproductive organs was unremarkable even after up to 13 weeks of exposure in males. All offspring were macroscopically ‘normal’, in particular no effects were seen on the external genitalia. Ano‑genital distance and male nipple count were not adversely affected by treatment. In the context of this study, the weight of evidence supports that 3-cyclohexene-1-carboxaldehyde multiconstituent has no effect on endocrine systems.  

 

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of test item for systemic toxicity was 12000 ppm (mean achieved doses of 714 mg/kg bw/day for males, 744 mg/kg bw/day for toxicity phase females, 761mg/kg bw/day for females during gestation and 1480 mg/kg bw/day for females during lactation).